scholarly journals Reconstitution of three-phase microtubule polymerisation dynamics

2016 ◽  
Author(s):  
Takashi Moriwaki ◽  
Gohta Goshima
Keyword(s):  
Growth Rate ◽  
Dynamic Instability ◽  
Molecular Level ◽  
The Other ◽  
Mitotic Kinase ◽  
The Core ◽  
Three Phase ◽  
Cytoplasmic Microtubules ◽  
Mitotic Cells

Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerisation cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, we reconstituted dynamic MT plus end behaviour involving all three phases, by mixing tubulin with five Drosophila proteins, EB1, XMAP215Msps, Sentin, kinesin-13Klp10A, and CLASPMast/Orbit. When singly mixed with tubulin, CLASPMast/Orbit strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASPMast/Orbit acted as an inducer of pausing. The mitotic kinase Plk1Polo modulated the activity of CLASPMast/Orbit and kinesin-13Klp10A, and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells, and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.

scholarly journals Five factors can reconstitute all three phases of microtubule polymerization dynamics

2016 ◽  
Vol 215 (3) ◽  
pp. 357-368 ◽  
Author(s):  
Takashi Moriwaki ◽  
Gohta Goshima
Keyword(s):  
Dynamic Instability ◽  
Molecular Level ◽  
The Other ◽  
Mitotic Kinase ◽  
The Core ◽  
Microtubule Polymerization ◽  
Five Factors ◽  
Cytoplasmic Microtubules ◽  
Three Phases

Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerization cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, we reconstituted dynamic MT plus end behavior involving all three phases by mixing tubulin with five Drosophila melanogaster proteins (EB1, XMAP215Msps, Sentin, kinesin-13Klp10A, and CLASPMast/Orbit). When singly mixed with tubulin, CLASPMast/Orbit strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASPMast/Orbit acted as an inducer of pausing. The mitotic kinase Plk1Polo modulated the activity of CLASPMast/Orbit and kinesin-13Klp10A and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.

scholarly journals Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable.

1988 ◽  
Vol 107 (3) ◽  
pp. 1025-1035 ◽  
Author(s):  
W Tao ◽  
R J Walter ◽  
M W Berns
Keyword(s):  
Dynamic Instability ◽  
The Other ◽  
Time Points ◽  
The Core ◽  
Interphase Cells ◽  
Cytoplasmic Microtubules ◽  
The Stability ◽  
Hydrolysis Of ◽  
Simultaneous Growth

To study the possible mechanism of microtubule turnover in interphase cells, we have used the 266-nm wavelength of a short-pulsed Nd/YAG laser to transect microtubules in situ in PtK2 cells at predefined regions. The regrowth and shrinkage of the transected microtubules have been examined by staining the treated cells with antitubulin mAb at various time points after laser irradiation. The results demonstrate that microtubules grow back into the transected zones individually; neither simultaneous growth nor shrinkage of all microtubules has been observed. The half-time of replacement of laser-dissociated microtubules is observed to be approximately 10 min. On the other hand, exposure of the core of the microtubule, which is expected to consist almost completely of GDP-tubulin, by transecting the internal regions of the microtubule does not render the remaining polymer catastrophically disassembled, and most transected microtubules with free minus ends do not quickly disappear. Taken together, these results suggest that most microtubules in cultured interphase cells exhibit some properties of dynamic instability (individual regrowth or shrinkage); however, other factors in addition to the hydrolysis of GTP-tubulin need to be involved in modulating the dynamics and the stability of these cytoplasmic microtubules.

Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardi

1986 ◽  
Vol 6 (2) ◽  
pp. 462-469
Author(s):  
S Merchant ◽  
L Bogorad
Keyword(s):  
Molecular Level ◽  
The Other ◽  
Mature Protein ◽  
Cytochrome C552 ◽  
Translatable Mrna ◽  
Electron Carriers ◽  
Different Levels ◽  
Polyadenylated Mrna ◽  
Photosynthetic Electron Transfer

Plastocyanin and cytochrome c552 are interchangeable electron carriers in the photosynthetic electron transfer chains of some cyanobacteria and green algae (P. M. Wood, Eur. J. Biochem. 87:9-19, 1978; G. Sandmann et al., Arch. Microbiol. 134:23-27, 1983). Chlamydomonas reinhardi cells respond to the availability of copper in the medium and accordingly accumulate either plastocyanin (if copper is available) or cytochrome c552 (if copper is not available). The response occurs in both heterotrophically and phototrophically grown cells. We have studied the molecular level at which this response occurs. No immunoreactive polypeptide is detectable under conditions where the mature protein is not spectroscopically detectable. Both plastocyanin and cytochrome c552 appear to be translated (in vitro) from polyadenylated mRNA as precursors of higher molecular weight. RNA was isolated from cells grown either under conditions favorable for the accumulation of plastocyanin (medium with Cu2+) or for the accumulation of cytochrome c552 (without Cu2+ added to the medium). Translatable mRNA for preapoplastocyanin was detected in both RNA preparations, although mature plastocyanin was detected in C. reinhardi cells only when copper was added to the culture. Translatable mRNA for preapocytochrome, on the other hand, was detected only in cells grown under conditions where cytochrome c552 accumulates (i.e., in the absence of copper). We conclude that copper-mediated regulation of plastocyanin and cytochrome c552 accumulation is effected at different levels, the former at the level of stable protein and the latter at the level of stable mRNA.

Gene Disruption Studies of Penicillin-Binding Proteins 1a, 1b, and 2a in Streptococcus pneumoniae

1999 ◽  
Vol 181 (20) ◽  
pp. 6552-6555 ◽  
Author(s):  
JoAnn Hoskins ◽  
Patti Matsushima ◽  
Deborah L. Mullen ◽  
Joseph Tang ◽  
Genshi Zhao ◽  
...  
Keyword(s):  
Growth Rate ◽  
Streptococcus Pneumoniae ◽  
Gene Disruption ◽  
Binding Proteins ◽  
The Other ◽  
Penicillin Binding Protein ◽  
Disruption Mutant ◽  
Penicillin Binding Proteins ◽  
Genes Encoding

ABSTRACT The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a inStreptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2amutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, andpbp2a genes are dispensable but that eitherpbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.

scholarly journals Yeast Bim1p Promotes the G1-specific Dynamics of Microtubules

1999 ◽  
Vol 145 (5) ◽  
pp. 993-1007 ◽  
Author(s):  
Jennifer S. Tirnauer ◽  
Eileen O'Toole ◽  
Lisbeth Berrueta ◽  
Barbara E. Bierer ◽  
David Pellman
Keyword(s):  
Cell Cycle ◽  
Dynamic Instability ◽  
Microtubule Dynamics ◽  
Yeast Protein ◽  
Gene Encoding ◽  
Cytoplasmic Microtubule ◽  
A Cell ◽  
Cytoplasmic Microtubules

Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells. Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1. During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state. Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein. Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules. This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end. Our in vivo data provide evidence of a cell cycle–specific microtubule-binding protein that promotes microtubule dynamicity.

scholarly journals Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardi.

1986 ◽  
Vol 6 (2) ◽  
pp. 462-469 ◽  
Author(s):  
S Merchant ◽  
L Bogorad
Keyword(s):  
Molecular Level ◽  
The Other ◽  
Mature Protein ◽  
Cytochrome C552 ◽  
Translatable Mrna ◽  
Electron Carriers ◽  
Different Levels ◽  
Polyadenylated Mrna ◽  
Photosynthetic Electron Transfer

Plastocyanin and cytochrome c552 are interchangeable electron carriers in the photosynthetic electron transfer chains of some cyanobacteria and green algae (P. M. Wood, Eur. J. Biochem. 87:9-19, 1978; G. Sandmann et al., Arch. Microbiol. 134:23-27, 1983). Chlamydomonas reinhardi cells respond to the availability of copper in the medium and accordingly accumulate either plastocyanin (if copper is available) or cytochrome c552 (if copper is not available). The response occurs in both heterotrophically and phototrophically grown cells. We have studied the molecular level at which this response occurs. No immunoreactive polypeptide is detectable under conditions where the mature protein is not spectroscopically detectable. Both plastocyanin and cytochrome c552 appear to be translated (in vitro) from polyadenylated mRNA as precursors of higher molecular weight. RNA was isolated from cells grown either under conditions favorable for the accumulation of plastocyanin (medium with Cu2+) or for the accumulation of cytochrome c552 (without Cu2+ added to the medium). Translatable mRNA for preapoplastocyanin was detected in both RNA preparations, although mature plastocyanin was detected in C. reinhardi cells only when copper was added to the culture. Translatable mRNA for preapocytochrome, on the other hand, was detected only in cells grown under conditions where cytochrome c552 accumulates (i.e., in the absence of copper). We conclude that copper-mediated regulation of plastocyanin and cytochrome c552 accumulation is effected at different levels, the former at the level of stable protein and the latter at the level of stable mRNA.

In vitro studies on the induction of sporogenous tissue on leaves of cinnamon fern. II. Some aspects of carbohydrate metabolism

1973 ◽  
Vol 51 (2) ◽  
pp. 341-349
Author(s):  
William H. Harvey ◽  
James D. Caponetti
Keyword(s):  
Growth Rate ◽  
The Other ◽  
Gas Liquid Chromatography ◽  
Initial Part ◽  
Sporogenous Tissue ◽  
Storage Carbohydrate ◽  
Active Substrate ◽  
Carbohydrate Levels ◽  
Endogenous Carbohydrates

Since previous studies indicated a requirement for high levels of exogenous sucrose for induction of sporogenous tissue on excised set III leaves of cinnamon fern, the initial part of this study was performed to determine which hexose moiety of sucrose would serve as the more active substrate for sporangial induction. Glucose was found to be preferentially used for sporophyll induction and resulted in more extensive development than was seen in those leaves grown on fructose. Endogenous carbohydrate levels were measured by gas–liquid chromatography. In decreasing order of quantity, freshly excised primordia contained xylans, sucrose, fructose, β-D-glucose, xylose, and α-D-glucose. In leaves cultured on medium with sucrose levels of 2, 4, 6, 8, and 10%, the highest levels of all endogenous carbohydrates studied were found in leaves grown on medium with 2% sucrose. The xylans accumulated in leaves grown on medium with all sucrose levels and the amounts present were generally above those of uncultured primordia. The xylans represented the highest quantities of the carbohydrates measured and appeared to be a major storage carbohydrate in both cultured and uncultured leaves. The concentrations of sucrose, on the other hand, generally declined and were, in all cases, below the amounts in uncultured primordia. Increasing levels of exogenous sucrose above 2% generally resulted in a decline in levels of the other internal carbohydrates after [Formula: see text] weeks of culture, but the respective quantities were generally above those of freshly excised primordia. Enzymatic analysis failed to reveal any starch in the leaves. It is suggested that although increasing sucrose levels resulted in an increase in the growth rate, it would appear that sucrose is important in sporangial induction because of some mechanism in addition to its effect on an increasing growth rate.

scholarly journals 3.7 Å cryo-EM structure of the core Centromere Binding Factor 3 complex

2017 ◽  
Author(s):  
Wenjuan Zhang ◽  
Natalya Lukoynova ◽  
Shomon Miah ◽  
Cara K Vaughan
Keyword(s):  
Atomic Structure ◽  
The Other ◽  
In Vitro Assays ◽  
Core Complex ◽  
The Third ◽  
The Core ◽  
Binding Factor

SummaryThe Centromere Binding Factor 3 (CBF3) complex binds the third Centromere DNA Element in organisms with point centromeres, such as S. cerevisiae. It is the only essential centromere binding complex as it facilitates genetic specification of point centromeres. It is therefore the most fundamental complex of the kinetochore in these organisms and its association with centromere DNA allows association of all other kinetochore components. We have determined the atomic structure of the core complex of CBF3, comprising 3 of its 4 components, using cryo-EM. The architecture of the complex is 'U'-shaped, with a deep, strongly basic channel that is narrow at one end and wide at the other. Combining our structure and in vitro assays, we present a model for its association with centromere DNA.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

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